Nature - USA (2020-01-16)

(Antfer) #1

Methods


Data reporting
No statistical methods were used to predetermine sample size. The
experiments were not randomized and investigators were not blinded
to allocation.


TT-OAD2 synthesis
Several azoanthracene-based derivatives are reported as potent
agonists of the GLP-1R (WO10114824), and a compound from this
series known as OAD2 was selected for our studies (WO14113357).
OAD2, (S)-2-{[(3S,8S)-3-[4-(3,4-dichloro-benzyloxy)-phenyl]-7-((S)-1-
phenyl-propyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxino[2,3-g]isoquinoline-
8-carbonyl]-amino}-3-[4-(2,3-dimethyl-pyridin-4-yl)-phenyl]-propionic
acid, was synthesized using procedures previously described (see exam-
ple 179 in WO10114824), and a dihydrochloride salt form (OAD2.2HCl)
was prepared by standard methods from the free base. Therefore, TT-
OAD2 is the dihydrochloride salt of OAD2 in patent WO14113357. The
purity of TT-OAD2 was determined by liquid chromatography–mass
spectrometry (LC–MS) to be 98.62%.


Constructs
GLP-1R was modified to contain either a 2xcMyc-N-terminal epitope tag
(for signalling and radioligand-binding assays) or a Nanoluc tag (with
a 12xGly linker; for NanoBRET binding studies) after the native signal
peptide. For β-arrestin recruitment assays, a C-terminal Rluc8 was fused
to the C terminus of the receptor. For G-protein conformational assays,
a Nanoluc flanked by SGGGGS linkers was inserted into Gαs and Gαi2
after G(h1ha10) in Gαs or E(HA.03) in Gαi2 as previously described^30 ,^31.
These were used in conjunction with an N-terminally Nluc-labelled Gγ 2.
For G-protein steady-state assays, G-protein NanoBit-split luciferase
constructs were generated by fusing the LgBIT after G(h1ha10) in Gαs
or E(HA.29) in Gαi2 and the SmBIT to Gγ2. For structural studies, human
GLP-1R in the pFastBac vector was modified to include an N-terminal
Flag tag epitope and a C-terminal 8×histidine tag; both tags are remov-
able by 3C protease cleavage. These modifications did not alter the
pharmacology of the receptor^3. A dominant-negative Gαs construct
was generated previously by site directed mutagenesis to incorporate
mutations that alter nucleotide handling, stabilize the G 0 state and
interactions with the βγ subunits^30.


Insect cell expression
GLP-1R, human dominant-negative Gαs, His6-tagged human Gβ 1 and Gγ 2
were expressed in Tni insect cells (Expression systems) using baculovi-
rus. Cell cultures were grown in ESF 921 serum-free media (Expression
Systems) to a density of 4 million cells per ml and then infected with
three separate baculoviruses at a ratio of 2:2:1 for GLP-1R, dominant-
negative Gαs and Gβ 1 γ 2. Cells were obtained by centrifugation 60 h
after infection and the cell pellet was stored at −80 °C.


Purification of the TT-OAD2–GLP-1R–Gs complex
Cell pellet was thawed in 20 mM HEPES, pH 7.4, 50 mM NaCl, 2 mM
MgCl 2 supplemented with cOmplete Protease Inhibitor Cocktail tablets
(Roche). Complex formation was initiated by addition of 50 μM TT-
OAD2, Nb35–His (10 μg ml−1) and apyrase (25 mU ml−1, NEB) to catalyse
hydrolysis of unbound GDP and allow for stabilization of the G 0 state;
the suspension was incubated for 1 h at room temperature. Membrane
was solubilized by 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG,
Anatrace) supplemented with 0.3% (w/v) cholesteryl hemisuccinate
(CHS, Anatrace) for 2 h at 4 °C. Insoluble material was removed by
centrifugation at 30,000g for 30 min and the solubilized complex
was immobilized by batch binding to M1 anti-Flag affinity resin in the
presence of 3 mM CaCl 2. The resin was packed into a glass column and
washed with 20 column volumes of 20 mM HEPES pH 7.4, 100 mM NaCl,
2 mM MgCl 2 , 3 mM CaCl 2 , 1 μM OAD, 0.01% (w/v) MNG and 0.006% (w/v)


CHS before bound material was eluted in buffer containing 5 mM EGTA
and 0.1 mg ml−1 Flag peptide. The complex was then concentrated using
an Amicon Ultra Centrifugal Filter (molecular mass cut off 100 kDa)
and subjected to size-exclusion chromatography on a Superdex 200
Increase 10/300 column (GE Healthcare) that was pre-equilibrated
with 20 mM HEPES pH 7.4, 100 mM NaCl, 2 mM MgCl 2 , 1 μM OAD, 0.01%
(w/v) MNG and 0.006% (w/v) CHS to separate complex from contami-
nants. Eluted fractions consisting of receptor and G-protein complex
were pooled and concentrated. Final yield of purified complex was
approximately 0.15 mg per litre of insect cell culture.

Electron microscopy
Samples (3 μl) were applied to a glow-discharged Quantifoil R1.2/1.3
CuRh 200 mesh holey carbon grid (Quantifoil GmbH) and were flash
frozen in liquid ethane using the Vitrobot mark IV (Thermo Fisher
Scientific) set at 100% humidity and 4 °C for the prep chamber. Data
were collected on a Titan Krios microscope (Thermo Fisher Scientific)
operated at an accelerating voltage of 300 kV with a 50 μm C2 aperture
at an indicated magnification of 105 K in nanoprobe EFTEM mode.
Gatan K3 direct electron detector positioned post a Gatan Quantum
energy filter, operated in a zero-energy-loss mode with a slit width
of 25 eV was used to acquire dose fractionated images of the GLP-1R
TT-OAD2-bound sample without an objective aperture. Movies were
recorded in hardware-binned mode (previously called counted mode
on the K2 camera) yielding a physical pixel size of 0.826 Å pixel−1 with
an exposure time of 3.715 s amounting to a total dose of 65.6 e− Å−2 at a
dose rate of 12.2 e− pixel−1 s−1, which was fractionated into 62 subframes.
A second dataset of 1,568 micrographs was also recorded using the
same microscope but in ‘super-resolution’ mode on the K3 detector,
the physical pixel size was 0.413 Å with an exposure time of 4.015 s
amounting to a total dose of 63.5 e− Å−2, which was fractionated into 67
subframes. Defocus range was set between −0.7 and −1.5 μm. A total of
3,158 plus 1,568 movies were collected in two data collection sessions.

Electron microscopy data processing
Movies were motion-corrected with UCSF MotionCor2^32 (movies col-
lected in super-resolution mode were Fourier scaled by a factor of ×2
to match the pixel size of the larger data set). This was followed by CTF
estimation sing the GCTF software packag^33. Particles were picked
from the micrographs using the automated reference-free procedure
in RELION^34 ,^35. Reference free 2D and 3D classification (by generating
multiple ab initio models with no structural identity enforced) was
carried out in CryoSPARC (v.2.5.0)^36. A homogeneous subset of par-
ticles was then subjected to movie refinement and Bayesian particle
polishing as implemented in RELION (v.3.0). This homogeneous subset
of polished particles was used in a 3D refinement in RELION and then
further classified into 3D classes with alignment of Euler angles not
taken into account. Particles belonging to the 3D class that yielded
the best resolved map were then subjected to signal subtraction to
subtract density due to the detergent micelle and the alpha domain
of the G protein. Final 3D refinement was performed in RELION (3.0)
yielded a map of resolutions 3.01 Å. Local resolution estimations were
performed using the ResMAP software packag^37.

Atomic model refinement
Fitting the model to the cryoEM electron density map was achieved
using the MDFF routine in namd^38. The fitted model was further refined
by rounds of manual model building in coot^39 and real space refinement
as implemented in the Phenix software package^40 , the model restraints
for the TT ligand were prepared by using the coordinates generated
from Chem3D and the ELBOW software package^41. The ligands were
fitted after the first round of real-space refinements, manually first in
coot^39 , then refined using Phenix real-space refinement^42. Ramachan-
dran, rotamer and secondary structure restraints were applied for the
first round of real-space refinement, and after manual inspection and
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